Using intravital 2-photon imaging methods developed in the LBS over the past several years, we are now able to routinely image peripheral organs and tissues such as the liver, kidney, bone marrow, gut, and skin so that immune effector cell behavior and to some extent effector functions during infectious processes can be observed. Using these new methods, we previously described lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver, the differences in mobility between myeloid and lymphoid components of these granulomas, and the specific changes in granuloma structure that accompany anti-TNF treatment. During the past year, we have extended these studies to compare the local effector responses of antigen-specific and unspecific CD4+ T cells within BCG-induced granulomas and to relate the migration properties of the T cells to their effector activity. These data show that only a small fraction of the antigen-specific T cells within a granuloma undergo migration arrest at any time and that the small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma, as assessed by isolation and intracellular staining of the cells from the infected liver. Confocal imaging data show that these T cells polarize their secretion of the effector cytokine towards sites of antigen load (bacteria). The limited migration arrest of antigen-specific T cells and the low number of cytokine secreting cells making just detectable levels of IFNgamma is not an artifact of the analytic method, because injection of high levels of the cognate antigenic peptide into the infected animals results in both migration arrest of nearly all the T cells and production of 1-2 logs more IFNgamma per cell by 80-90% of the T cells under these conditions. These data suggest that during normal immune responses to mycobacteria in liver granulomas, there is very limited antigen presentation just sufficient at any moment to activate a small fraction of all available effector cells into a cytokine-secretory state, and to do so just at the margin of quantitative response potential. These findings provide entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. Many of these observations have been repeated in M tuberculosis-infected animals, arguing that it is not the low pathogenicity of the BCG that leads to such limited effector responses, or the differential dynamic behavior of macrophages or T cells within granulomas. The finding that only a small fraction of the cells being imaged in a tissue are actually engaged in effector function at any time and that these have a dynamic behavior distinct from the bulk of the imaged cells raises critical questions about many existing and ongoing studies in other laboratories using intravital 2-photon imaging, in which the bulk or average behavior of clonally-related cells is taken as representative of the functional population;our data suggest this is a very dangerous way to interpret these images and that in many cases, investigators may be missing the distinct behavior of a small percent of the cells that contribute all or most of the relevant biological activity in the system under study. In addition to the granuloma model, we are presently undertaking related imaging studies of infected animals involving influenza , mycobacteria, and P aeruginosa in the lungs as well as S aureus, especially MRSA strains, in the skin. The aim is to determine what aspects of immune cell behavior are common to different infections are different tissue sites, and which are distinct, as well as how these differences relate to the efficacy of host defense. We have undertaken a detailed analysis of innate cell (neutrophil, macrophage, and dendritic cell) motility in 3 dimensional artificial environments collaborators have prepared using microfabrication methods to allow precise control of substrate porosity, rigidity, and chemokine gradients, to obtain data suitable for computational modeling and mechanistic examination of the modes of immune cell locomotion. Related studies are also being undertaken with these same cells in real tissue environments, to compare the artificial 3D data to those obtained in tissues and to develop a deeper understanding of how the migratory activities of these cells are controlled and how effective scanning for infectious agents takes place. We are also evaluating the effect of viral and other infections on the state and function of non-hematopoietic cells (fibroblastic reticular cells and other stromal elements) in secondary lymphoid tissues. During the past year we have developed new staining protocols that permit as many as 8 colors to be used to detail the distribution of stoma cells subsets and immune (hematopoietic) cells within lymphoid tissues, and begun to assess using 2 photon imaging the effects of LCMV infection on the stromal components of lymph nodes and spleen, along with concomitant changes in chemokine expression and T cell migration.